A virulent form of bloody diarrhea is caused by the Enterohemorrhagic Escherichia coli (EHEC). This pathogen is the most common infectious cause of bloody diarrhea (also called hemorrhagic colitis [HC]) in the United States (Centers for Disease Control and Prevention (executive summary). MMWR 43(No.RR-5):1-18 (1994); Griffin, P. M. et al. Annals of Internal Med. 109:705 (1988)). One serotype in particular, 0157:H7, is the most commonly isolated serotype of EHEC in the United States, and has been linked to a significant number of outbreaks of HC beginning in 1982 (Riley, L. W. et al. N.Eng.J. Med. 308:681 (1983)).
The primary mode of transmission of EHEC occurs through ingestion of contaminated food, particularly undercooked hamburger (Doyle, M. P. and Schoeni, J. L. Appl. Environ. Microbiol. 53:2394 (1987); Samadpour, M. et al. Appl. Environ. Microbiol. 60:1038 (1994)). Among people infected by EHEC, as many as 5% suffer a serious complication called Hemolytic Uremic Syndrome (HUS), a condition caused by the action of Shiga-like toxins that target and destroy cells lining blood vessels (endothelial cells), such as those present in the glomeruli of the kidney. (Johnson, W. M. et al. Lancet. i:76 (1983); O'Brien, A. D. et al. Lancet. i:702 (1983)). HUS can result in permanent kidney damage or even complete kidney failure.
Although EHEC can cause very serious illness even in healthy adults, young children in particular are at greater risk of dying or suffering permanent damage from the infection. Others for whom the infection can be particularly dangerous include the elderly and the immuno-compromised. With the prevalence of EHEC in cattle and the subjective nature of differentiating between cooked and undercooked hamburger, a convenient stop at a fast food restaurant, or even a family barbecue, can result in family tragedy.
One key to the deadly nature of EHEC is the bacteria's ability to produce attaching/effacing (A/E) intestinal lesions in the colon, such as those demonstrated in gnotobiotic pigs (Tzipori, S. et al. Infect. Immun. 57:1142 (1989)). The A/E lesions demonstrated in pigs are characterized by intimate bacterial adherence to the mucosal cells of the intestinal lining and dissolution of microvilli (McKee, M. L. et al. Infect. Immun. 63:3739 (1995); Tzipori, S. et al. Infect. Immun. 57:1142 (1989)). Similar lesions have been seen in human laryngeal epithelial (HEp-2)(ATCC # CCL23) cells in tissue culture (McKee, M. L. et al. Infect. Immun. 63:3739 (1995); Tzipori, S. et al. Infect. Immun. 57:1142 (1989)).
In 1990, Jerse et al. identified a chromosomal gene in a related diarrheagenic E. coli strain, Enteropathogenic E. coli (EPEC). That gene, designated eae, was found to be required for the bacterium to produce A/E lesions in tissue culture (Jerse, A. E. et al. Proc. Natl. Acad. Sci. USA. 87:7839 (1990)). The eae gene encoded a 94 kDa outer membrane protein, called Eae, which is the intimin of EPEC. A similar protein was demonstrated to be present in an EHEC 0157:H7 strain (Jerse, A. E. and Kaper, J. B. Infect. Immun. 59:4302 (1991)).
Recently, investigators demonstrated that intimin is necessary for adherence of EHEC to human epithelial laryngeal (HEp-2) cells and human ileocecal epithelial (HCT-8) cells (ATCC # CCL244) (McKee, M. L. et al. Infect. Immun. 63:3739 (1995)) and for formation of A/E lesions in the piglet intestine (Donnenberg, M. S. et al. J. Clin. Invest. 92:1418 (1993); McKee, M. L. et al. Infect. Immun. 63:3739 (1995)). Although human studies with EHEC have not been conducted, the intimin protein found in EPEC is strongly associated with the production of diarrhea and fever in human volunteers (Donnenberg, M. S. et al. J. Clin. Invest. 92:1412 (1993); Levine, M. M. et al. J. Infect. Dis. 152:550 (1985)).
Human volunteers (10 out of 10) challenged with EPEC strain E2348/69 mounted a notable immune response to the 94 kDa protein after 28 days (Levine et al. J Infect. Dis. 152:550, 1985)). In these human trials the only volunteer (1 out of 10) who failed to develop diarrhea after ingestion of E2348/69 was the individual in this group who had detectable antibody to the 94 kDa protein before challenge.
Two other bacterial species capable of inducing A/E lesions have been shown to contain the eae locus: Hafnia alvei (Albert, M. J. et al. J. Med. Microbiol. 37:310 (1992)) and Citrobacter freundii biotype 4280 (Schauer, D. B., and Falkow, S. Infect. Immun. 61:2486 (1993)). Although these bacteria are not generally associated with pathology in humans, they can cause significant disease in the animal species with which they are normally associated. For instance, Citrobacter freundii biotype 4280 is associated with gastrointestinal illness in mice. Mice often serve as control and test subjects in experiments. Costly and carefully controlled experiments can be jeopardized by an outbreak of this disease in an animal care facility. In addition, such bacterial species may become pathogenic to immuno-compromised patients, the young and the elderly.
Animals, such as cows, infected with bacterial strains expressing intimin may become ill themselves, in addition to serving as a source of such infections to others. Eradicating or even limiting these animal reservoirs of intimin-expressing bacteria in animals with antibiotic therapy would be prohibitively expensive. In addition, not only is antibiotic treatment of the infections in humans or animals costly, but the antibiotics themselves are associated with side effects that can be dangerous. As with EHEC, those side effects can be especially dangerous to young children and the elderly. Consequently, the need exists for another means of reducing the seriousness of the infections or preventing them altogether through promotion of protective immune responses against bacteria expressing intimin.
A further need is for forms of immunization that are less time consuming, expensive and painful than immunization through injection of antigens. Yet another need is for the generation of protective immune responses in the specific tissues involved at the point of infection, most often the gastrointestinal mucosa.
Other organisms infecting gastrointestinal tissue, including, but not limited to Salmonella sp. and Shigella sp., possess antigens against which an immune response could be generated. A need exists, however, for a means of targeting those antigens to gastrointestinal mucosa, in order to stimulate a mucosal immune response, as well as stimulating circulating antibodies.
Finally, a need exists for alternate means of delivering agents that promote a protective immune response.